R. solanacearum produces bacterial wilt worldwide in more than 200 plant species belonging to more than 50 botanical families, and many of these species susceptible to this pathogen are of agricultural interest. There are also many other crops that are colonized by the bacteria, but do not develop symptoms, which are considered tolerant crops. This bacterium especially attacks staple crops such as potatoes in more than eighty countries, with losses exceeding 950 million dollars. For this reason, it is considered as a potential agent of bioterrorism and in the European Union (EU) as a quarantine organism (Anonymous 2000: Council Directive 2000/29/EC), which is subject to strict prevention and control measures regulated by two European directives (Anonymous 1998, 2006: Council Directive 98/57/EC and Commission Directive 2006/63/EC).
R. solanacearum presents great intraspecific diversity, and has therefore long been considered a complex of species consisting of four phylogenetic groups or phylotypes. In 2014, after a taxonomic revision, a reclassification of this complex was proposed (Safni et al., 2014). The phylotypes I and III of R. solanacearum they have been classified as the new species R. pseudosolanacearum and phylotype IV in the new subspecies R. syzygii subsp. indonesiensis. Phylotype II retains the name of the species R. solanacearum. 
In the text of this document the term “the species formerly known as R. solanacearum” is used to refer to R. solanacearum in studies, data, patents, publications, literature, etc., prior to the taxonomic revision of Safni et al (2014), regardless of whether the name corresponds or not with the current classification. Furthermore, the term “R. solanacearum” is used to refer to the species R. solanacearum as described after the aforementioned taxonomic revision, i.e. the species is constituted solely by strains of phylotype II.
In Spain, as in most EU countries, there have been outbreaks of the disease caused by R. solanacearum in several areas, usually in potatoes and in some cases tomatoes. In all cases of outbreaks the eradication measures set out in the relevant legal regulations were implemented. However, this bacterium can survive in the environment, water, soil or other reservoirs (Alvarez et al., 2007, 2008, 2010). Thus, subsequent outbreaks have been associated with irrigation with surface water contaminated with this bacteria (Caruso et al., 2005), as these are one of the main routes of introduction and spread of the pathogen into new areas, which comes through irrigation water. In fact, it described the presence of R. solanacearum in various waterways both in Spain and virtually all EU countries. Therefore, the EU Directives prohibit irrigation with water contaminated with this bacteria.
This represents a practical problem for the farmer, as the most affected crops are irrigated crops (tomato and potato) and irrigation water is a scarce commodity in Spain and other countries of the Mediterranean basin, where the bacteria are also present. Also in the area where outbreaks of the disease have been detected, host plants cannot be grown over a period of at least 4 years from the detection of an outbreak.
The large capacity of survival of R. solanacearum in the environment (water, soil or other reservoirs) impedes control. In the past and up to the present moment, control through farming practices and chemical control are being used. Thus, in areas of the world where this pathogen is present in the soil, usually in developing countries, growing host plants is problematic and the most widely used methods of control are farming techniques with varying results. Resistant cultivars have also been obtained, however, resistance may be unstable (Hartman and Elphinstone, 1994).
With respect to control via chemical or physical treatments, generally they are not effective. The application of copper compounds, antibiotics and soil fumigants have been used without much success (Lopez and Biosca, 2005), and are also expensive and have a great impact on the environment. Other chemical treatments such as chlorination or physical treatments, such as ultraviolet radiation of water contaminated with bacterial pathogens, have limited effectiveness where particles are present in the water, even if very small (Marco-Noales et al., 2008). Furthermore, it has been shown that both of aforementioned disinfectant treatments can induce various bacteria to enter into the viable but non-culturable state (VBNC) (Oliver et al., 2005 Santander et al, 2012.) Or cause reversible cell damage (McFeters and LeChevallier, 2000). These bacterial cells in the VBNC state or having reversible damage can recover cultivability and pathogenicity after coming into contact with susceptible host plants (Santander et al., 2012). These circumstances demonstrate the need for alternative methods that preferably destroy the bacterial cells.
An alternative would be biological control using specific bacteriophages of R. solanacearum. This biocontrol strategy has been successful in the treatment of other diseases caused by phytopathogenic bacteria (Jones et al., 2007).
In that vein, Japanese patent JP4532959-B2 (publication number JP2005278513) describes three types of bacteriophages with bacteriolytic activity on Japanese strains of the species formerly known as R. solanacearum and from 2014 belonging to the new species R. pseudosolanacearum. Type 1, with double-stranded DNA genome (dsDNA) of approximately 250 kbp, and types 2 and 3, with a genome of single-stranded DNA (ssDNA) of 4.5 and 6 kbp respectively. Bacteriophages are characterized and distinguished by the size of their genomes and their activity against six strains of bacteria (C319, M4S, Ps29, Ps65, Ps72 and Ps74), all of which are sensitive to type 1 bacteriophages, while the type 2 only lyses one strain (the C319 strain) and type 3 lyses four of the six strains (M4s, Ps29, PS65 and Ps74) and a fifth in one trial (the C319 strain). Trials with restriction endonucleases have only been performed with type 1 bacteriophages and show that its genome (dsDNA) targets PstI and KpnI, since in the resulting restriction profiles several distinct bands are observed. Finally, the use of two of the three types of bacteriophages (types 1 and 2) to control the disease caused by species formerly known as Ralstonia solanacearum (currently R. pseudosolanacearum) by addition to soil cultivation where the plant to be protected is growing.
Japanese Patent JP4862154-B2 arises from a limitation of the above because, as is indicated therein, “it is not enough to control the effect.” In this second patent is included a new type of bacteriophage lytic activity against all strains of the species formerly known as R. solanacearum which were tested (only a total of 15 strains) and perform characterization, but do not demonstrate their capacity for biocontrol, since no biocontrol tests were performed on plants with this new type of bacteriophage. In short, the second patent only provides a new type of lytic bacteriophage with a range of hosts that is seemingly larger than previous types.
Yamada et al (2007) describe the isolation of four types of bacteriophages that infect specific strains of the species formerly known as R. solanacearum, from 2014 belonging to the new species R. pseudosolanacearum, from soil samples taken in different areas Japan. These authors perform structural characterization including a morphological characterization by electron microscopy virions and molecular characterization by restriction analysis of the four types of bacteriophages, and some lytic activity tests with cultured bacteria in Petri dishes. Two types of bacteriophages described are mioviruses, the genus to which all the bacteriophages infecting the species formerly known as R. solanacearum (now R. pseudosolanacearum) belong described before publication of the article by Yamada et al, and the other two types are filamentous bacteriophages of the inovirus type. Among other applications, the usefulness of the bacteriophages' lytic activity as biocontrol agents for the eradication of the species formerly known as R. solanacearum (now R. pseudosolanacearum) in contaminated soils and preventing wilting caused by the bacteria in vegetable crops is suggested, showing preference for bacteriophages capable of infecting a wide range of pathogenic strains, although only testing 15 strains of this host. The only tests with plants included in the aforementioned article referred to observing a plant injected with a strain previously infected with one of the isolated lysogenic bacteriophages, concluding that filamentous phages were not satisfactory for a disease control effect. This same test method has been used in other studies of the same group also with filamentous bacteriophages of the Inoviridae family [see Addy et al., 2012 and International Patent Application WO2012/147928], where it is shown that aforementioned method of inoculation, in the case of lysogenic bacteriophage type ΦRSM, leads to the emergence of avirulent strains of R. solanacearum that help control the disease caused by the virulent forms of aforementioned bacteria. However, besides the fact that the inoculation tests have only been performed in 5 plants per strain and without repetition [see material and methods Addy et al., 2012], the practical application of this test method in nursery or in the field is highly questionable because the work required to inject each individual plant.
In another article by the same research group (Fujiwara et al., 2011) test results demonstrating utility as biocontrol agents on the species formerly known as R. solanacearum (currently R. pseudosolanacearum) are provided for two types of bacteriophages of the Myoviridae family described by Yamada and collaborators (2007, 2010), ΦRSA1 and ΦRSL1, as well as the effect of other additional bacteriophage ΦRSB1, with was previously isolated (Kawasaki et al., 2009). The latter belongs to the Podoviridae family and, like ΦRSA1, is capable of causing lysis in a higher number of strains, up to 13 of 15 strains of the species formerly known as R. solanacearum (of which at least 13 are currently classified as R. pseudosolanacearum) (Yamada et al., 2007; Kawasaki et al, 2009). It is shown that treatment of the bacteria with ΦRSA1, ΦRSB1 and ΦRSL1 either individually or in possible combinations, except treatment with ΦRSL1 alone, results in a rapid decrease in cell density of the host bacteria, which is only an initial decrease because is followed by the appearance of resistance visible by OD (optical density) in less than 2 days. To avoid such resistance, Fujiwara and colleagues (2011) selected the use of miovirus ΦRSL1 individually with respect to other combinations with ΦRSA1 and ΦRSB1, although it is noteworthy that this is the lowest bacteriophage lytic potential of the three.
Tests performed on plants described in the article by Fujiwara et al (2011), all done with ΦRSL1, entailed two treatments with a suspension of aforementioned bacteriophage at high concentrations conducted with a spacing of one month, first to the seed and then to the resulting plants; after two days these plants were individually inoculated with the bacterial suspension by direct contact to the cut tips of the roots (for 30 seconds) and then transplanted. This method of inoculation, like the previous used by these authors in other publications and patents, it is very difficult if not impossible to implement in nurseries or in cultivated fields. Furthermore, in aforementioned publication the reproducibility of the results is not indicated.
Fujiwara et al (2011) also describe stability tests on ΦRSL1 on two plants pre-treated with aforementioned bacteriophage and soil in contact therewith, detecting bacteriophages in the roots and in the rhizosphere soil 4 months after inoculation, although it was not verified whether the recovered bacteriophages are of the same type as those inoculated. Also, the effect of temperature on the stability of ΦRSA1, ΦRSB1 and ΦRSL1 was tested in presence and absence of soil (in SM buffer, Tris-HCl, NaCl, MgSO4 and gelatine). The stability was monitored for only 15 days. At the same temperature, major differences in the stability of the three bacteriophages in the presence of soil were observed. Both in the absence and presence of soil, the differences were greater with increasing temperature but, in the absence of soil, marked differences start from 28° C. After 15 days incubation in the absence of soil at 50° C., 10% of ΦRSL1 bacteriophages survived, ΦRSB1 bacteriophages were not detected after 9 days of incubation, and after 3 days of incubation in the case of ΦRSA1.
Thus, the work of the Japanese group which includes Yamada, Addy and Fujiwara, show that in some cases and with some bacteriophages that infect the species formerly known as R. solanacearum (now R. pseudosolanacearum), can be used as agents for prevention of disease caused by the bacteria. Except in one case, the podovirus ΦRSB1, ruled out by these authors for biocontrol (Fujiwara et al., 2011), bacteriophages described and used in aforementioned tests belong to families Myoviridae or Inoviridae. In aforementioned tests, the bacteriophages are applied directly to the soil, seedlings or in roots or stems of plants. No evidence of the potential effectiveness of other means of administration of bacteriophages are given, such as perhaps through irrigation water. This is not surprising, because in Japan R. pseudosolanacearum has not been detected in natural watercourses, unlike the case in many European countries and in some areas of the US where this type of water is one of the reservoirs and routes of dissemination of R. solanacearum. 
The ability to control R. solanacearum in water by using bacteriophages has been seen in other studies in some areas of eastern and western Europe. Thus, the summary of the Georgian patent application GEU20041089 suggests neutralization of aforementioned bacteria in plants, soil and water using a mixture of polyvalent bacteriophages in equal proportion.
The present group of inventors, however, has previously suggested the use of specific bacteriophages of the species formerly known as R. solanacearum in irrigation water to control bacterial wilt caused by aforementioned pathogen (Alvarez et al., 2006a; Alvarez et al, 2006b). The influence of the conditions of watercourses in the survival of the bacteria have been verified, finding that the presence of native microbiota and temperatures of 24° C. favoured the disappearance with respect to the temperature of 14° C. and sterile water used as a control (Alvarez et al., 2006a). None of these disclosures provides information about the phylotype of the tested strains, so it cannot be aforementioned that these phages could act on the current R. solanacearum, consisting of strains phylotype II.
The group of the present inventors have also reported the isolation of specific lytic bacteriophages of the species formerly known as R. solanacearum from rivers in Spain (Alvarez et al., 2006a, Alvarez et al., 2006b), but without specifying the method of isolation and the specific place of isolation of each. The authors have reported initial data on the characterization of one of them, saying that it seems to show lytic activity between 14° C. and 31° C., but not at lower temperatures (9° C.) or higher temperatures (32-39° C.) even in natural irrigation water pH ranges from 6.5 to 8.2. The initially characterized bacteriophage appears to be specific to the species formerly known as R. solanacearum, it shows lytic activity on 30 strains of different origins, of which the phylotype has not been disclosed. The bacteriophage showed lytic activity against other bacterial isolates of river water. Aforementioned bacteriophage also results in the reduction of bacterial wilt in tomato plants irrigated with water containing mixtures of bacteriophage and the species formerly known as R. solanacearum. 
The data obtained by Alvarez and his colleagues support the possibility of using bacteriophages to prevent and/or control bacterial wilt, in particular by addition to irrigation water. However, data released by the group so far do not identify the specific watercourses from which the lytic bacteriophages obtained by them can be isolated. Nor was specific data given on any of aforementioned bacteriophages in order to facilitate the identification via the structural characteristics. Data on the range of temperatures and pH in which aforementioned bacteriophages are active has only been reported for one, which was also claimed to be able to lyse 30 different strains of the species formerly known as R. solanacearum, without specifying the particular strains in which it has proven activity.
Therefore, there is still a lack of specific lytic bacteriophages which have been established to, individually, be able to reduce bacterial wilt when added to irrigation water. For the single bacteriophage on which this type of tests have been carried out, reflected in the academic presentation made by the present group of inventors (Alvarez et al, 2006a; Alvarez et al, 2006b), data have not been disclosed to allow identification other than by the lytic activity at different pHs and temperatures and the claim that decreases bacterial wilt in tomato plants, not revealing the genus or family or any concrete data on watercourses in which it is present (and from which it could be isolated) or the specific method of the isolation or the phylotype of the host strains that are affected.
Thus, neither for bacteriophages isolated by Alvarez et al. or for the bacteriophages whose use is suggested in the Georgian patent application GEU20041089 in the form of polyvalent mixtures, are there available data on survival under natural conditions, in particular on their survival in the conditions of the waters in which they would be used. A central factor in determining the suitability of a bacteriophage as biocontrol agent is precisely the survival under natural conditions. When bacteriophages of phytopathogenic are applied to soil or plants to eliminate aforementioned pathogenic bacteria, the time they can stay active until they find their target cell is the limiting factor in the process, which causes repeated applications of these bacteriophages to be required for effective control. And in the case of the species formerly known as R. solanacearum, its control in watercourses, especially in irrigation water and containers (tanks, storage vessels, reservoirs . . . ) it is important, especially in Europe, because:
i) the main crops affected by R. solanacearum are irrigated (especially potato and tomato),
ii) there is currently a shortage of water in Spain and other countries of the Mediterranean basin where the pathogen is present,
iii) there are official prohibitions throughout the EU to use contaminated water for irrigation of host plants (Anonymous, 1998, 2006 water: Council Directive 98/57/EC and Commission Directive 2006/63/EC),
iv) no control methods available for use in water,
v) a priority objective of EU policy is the conservation of the environment (Montesinos et al, 2008; Horizon 2020 Program).
Therefore, it would be desirable to control the populations of bacteria in river water and/or irrigation in a biological treatment that is effective and respectful of the natural environment. And, as was aforementioned previously, it is preferable for the method to result in the death of the bacteria.
In the case of plant pathogen R. solanacearum, the habitats of which are the host plants and soil, a biological agent that is supplied by water must have biological characteristics that allow it to survive in that environment, which is not the usual environment of the bacteria or its specific bacteriophages. If such survival is be prolonged and bacteriophages maintain their lytic activity on the host after long periods in water, this further favours applicability in the field as they can be transferred directly by natural and simple means such as water, without needing to encapsulated them or to provide other physical and/or biological means to protect their viability until contact with the target cell. This high survival rate would also facilitate the preparation of a commercial form, which could be in an aqueous medium without requiring refrigeration (or even lower temperatures) to maintain effectiveness.
However, it should be noted that bacteriophages are obligate intracellular parasites, and as such, require the host cell for perpetuation. Since they reach the cell in different ways, depending on the types of bacteriophages and types of host cells, survival time in the environment is expected in order to allow them to come into contact with the host cell. It is known that this time can vary between different bacteriophages, which requires study in each particular case. For example, significant variations are observed in the survival of bacteriophages the same serotype/genotype (Brion et al., 2002) or even between bacteriophages of aquatic pathogenic fish bacteria, the natural habitat of which is water (Pereira et al., 2011). In the latter case, three months has come to be regarding as good survival time of bacteriophages in water, accepting that bacteriophages displaying increased survival in water are good candidates for the control of bacterial fish diseases in aquiculture (Pereira et al., 2011).
It should be noted that R. solanacearum is a phytopathogenic bacterium whose natural environment is frequently the xylem of plants and soil, but not water. Since it is not an indigenous bacteria to aquatic environments, specific bacteriophages are not expected to have a high water survival rate. In fact, none of the previously mentioned publications and patents describes the viability and specific lytic activity of lytic bacteriophages of the species formerly known as Ralstonia solanacearum in environmental water in the absence of host cells.
And yet, it would be beneficial to have specific lytic bacteriophages against R. solanacearum, particularly that show a broad spectrum of strains of that species on which they are active and that also have a high survival rate in water, preferably of at least a month or more, more preferably several months. This could make possible biological treatment of irrigation water contaminated with R. solanacearum, using specific bacteriophages of the pathogen, which could prevent or reduce bacterial wilt in polluted areas that may impact susceptible plants. Treatment with these bacteriophages present the usual advantages over chemical treatments, as well as those associated with other treatments of biocontrol, especially with bacteriophages:
High specificity for the host cell,
Natural replication only in the host cell,
Harmless to other living beings, including microbiota beneficial to the plants to be protected,
Using biological treatment via irrigation water is easier and cheaper than chemical control, and does not require protective measures for staff during application, since it is harmless to humans,
Lower environmental impact, especially compared to copper compounds and antibiotics used in agriculture, for which many pathogens have developed resistance, reducing the effectiveness of such chemical treatments and also increasing the chemical contamination of soil, plants and fruit,
Less legal use restrictions, can be applied where chemical control is prohibited,
Since they are inert in their extracellular state, bacteriophages can be combined with other control strategies and/or biocontrol to increase disease control,
Easy, low-cost production because they increase their number in the presence of target bacteria,
Easy adjustment of the dose of bacteriophages to be used depending on the concentration of pathogen to be treated.
The present invention provides a solution to the problem of the absence of bacteriophages that display a broad spectrum of strains of the bacterium on which they are active and that also have a high survival rate in water, preferably at least one month, or more preferably, of at least several months.